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THE BACTERIOLOGY OF THE FEMALE 

REPRODUCTIVE ORGANS OF CATTLE 

AND ITS RELATION TO THE DISEASES 

OF CALVES 



A THESIS 

PRESENTED TO THE FACULTY OF THE GRADUATE SCHOOL 
OF CORNELL UNIVERSITY FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



BY 



CHARLES M.' CARPENTER 



Reprinted from the Annual Report of the New York State Veterinary College 
at Cornell University, Ithaca, N. Y., 1920-1921 



X-.M 



s 






>THE BACTERIOLGY OF THE FEMALE REPRODUCTIVE ORGANS OF 
f CATTLE AND ITS RELATION TO THE DISEASES OF CALVES 

Vr) Chaeles M. Cabpenteb 

5^ Department of Pathology and Bacteriology 

CyNew York State Veteiinary College at Cornell University, Ithaca, N. Y. 

~^}-^ Perhaps the most important diseases in animal pathology, or those receiv- 
(;^ng the most attention at least at the present time, are the pathological con- 
ditions associated with the breeding of cattle. From a study of the reproduc- 
' rtive efficiency in many large herds, Williajns (1) has found that it is about 
60 per cent of the ideal. The ideal which he considers normal and which 
herds, especially dairy herds, should strive to attain, is that each female 
in the herd should produce one living calf every twelve months, and that 
each heifer throw her first calf at two years of age. The U. S. Department 
of Agriculture placed the losses from observed contagious abortion at 
$20,000,000 for the year 1915 (2). A conservative estimate of all losses 
from infections associated with the breeding of cattle is 25 per cent, and 
since the valuation of all the cattle in the U. S., as reported January 1, 1921, 
is $1,934,185,000 (3) one can readily judge the enormous economic loss to 
the country. Moreover great losses are caused by these diseases in connec- 
tion with the work of improving the breeds and from a scientific point of 
view, by reason of the breaking up of experiments in heredity. 

The importance of the ))acterial content of the genital canal can easily be 
estimated from the foregoing statements. Much confusion has arisen in the 
past, and still exists, inasmuch as many observers in this field have studied 
the infections occurring in the diseased conditions without any knowledge 
of the microorganisms that may txist in the healthy reproductive organs of 
cattle. This is true of almost all the diseases that have been under close 
■ observation because it is primarily the abnormal condition that attracts the 
attention of the investigator. 

Because of the widely varied opinions concerning the different miccoorgan- 
isms existing in the reproductive (jrgans and their relation to the etiology of 
the diseases associated with them, it has seemed advisable to make a 
thorough bacteriological investigation of these organs, covering all ages and 
conditions, in an effort to improve the general conception of this subject 
and to correlate the facts with more accuracy. This work, started in the 
fall of 1917, has been carried on systematically and in great detail for nearly 
four years. In it I have tried to determine six things: First, what micro- 
organisms, if any, normally live in the organs comprising the female genital 
tract of cattle? Second, at what age of the animal and by what channels 
do the organisms enter the genital canal? Third, what relationship exists 
between these microorganisms and the pathological changes associated with 
them? Fourth, what effect is produced by these organisms on the fetus in 
the pregnant uterus, or on the calf at birth? Fifth, is there any evidence 
that bacteria which may have been transmitted to the calf from the dam lie 
dormant in the former until breeding age? Sixth, if these bacteria do persist 
until breeding age, is their habitat the genital tract or do they become localized 
elsewhere in the body thence proceeding to the reproductive organs st puberty 
and first pregnancy? 

History 
In reviewing the literature dealing with the bacteriology of the female 
genital tract of man and animals, one can find in human and veterinary medi- 
cine very little work which has been done upon these diseased tissues. As 
a result of the losses from puerperal fever in women, when aseptic methods 
were not understood and observed as carefully as they should have been, 
special attention was paid to the bacterial flora and chemical reaction of the 
vaginal secretions of pregnancy and non-pregnancy. The relation of the 
microorganisms found to the etiology of puerperal fever has been discussed at 
great length. Chief among the workers have been Doderlein (4), Williams 
(5), Kronig (6), Winter (7), Bensis (8), Bergholm (9), Walton and 



Medalia (10), Kiister (11) and Permar (12). These men did not agree 
entirely in their conclusions but in the main they found that in women the 
normal vaginal secretion is acid, as tested out with litmus paper, and because 
of this acidity very few bacteria are capable of living in the vagina. 
Doderlein reports that the acidity is due to a microorganism whicli lie lias 
described and which is called Doderlein's bacillus. He carried on experiments 
with this organism to prove the point but some of the other worl^ers did not 
agree with him. These men also stated that when the secretion contains large 
numbers of leucocytes, squamous epithelial cells, and bacteria, they consider 
it pathological. Under these conditions the reaction of tlie vaginal content 
approaches the neutrality point and in some cases it even becomes alkaline, 
in which case the bacteria are able to thrive. Kronig examined 300 cases and 
reported that the reaction of all these secretions was acid and tliat tliere were 
no streptococci present. He stated tliat this was the result with the patho- 
logical as well as with the normal secretions. Besides the gonococcus, the 
organisms found were for the most part streptococci, staphylococci, the 
thrush fungus (Oidium albicans) and occasionally a few rods. Sometimes 
they were found to be pathogenic for laboratory animals, but they often pro- 
duced no effect upon them and because of this fact some of the investigators 
considered the organisms to be in a state of low virulence. The streptoco- 
coc«us was thought to be most important because of its close relation to puer- 
peral fever. I have come across no systematic bacteriological studies on the 
flora of the upper part of the healthy genital canal of women, although some 
work has been done on the bacteriology of cervicitis, endometritis, salpingitis 
and ovaritis by Rosenow (13), Davis (14), Miller (15), Grad (16), Curtis 
(17) and Tweedy (18). 

In veterinary medicine even less work has been done on the bacterial flora 
of the reproductive organs of domestic animals than can be found in the 
literature having to do with human medicine. There has been an abundance 
of material, both normal and abnormal, contrary to the conditions found in 
human medicine, but no one has availed himself of the opportunity. Because 
of great losses of calves from various calf infections, some work has been 
done with the bacterial content of the vagina* of cattle, inasmuch as some of 
the early investigators believed that the calf became infected during the 
process of parturition. In 1904 Denzler (19) did considerable work on the 
bacteria commonly found in the vaginae of cattle and also some bacteriological 
work on the entire genital tract. He recorded staphylococci, streptococci and 
organisms from the colonaerogenes gi-oup in the vaginal vestibulum but stated 
that these bacteria are not able to grow within the healthy vagina. He con- 
sidered the external os uteri as the normal barrier between the germ-free 
zone in the genital canal and the zone which harbors bacteria, if, under 
abnormal conditions, the vagina contains bacteria. After a long series of 
experiments he concluded that the vaginae of calves, pregnant and non-preg- 
nant cows possess a power to disinfect themselves if microorganisms acci- 
dentally enter or are introduced therein. Experimentally he found this bac- 
tericidal action to l)c permanent, taking the tissues from 18 to 117 hours. He 
was of the opinion that the leucocytes, acting as phagocytes, and not the 
chemical reaction of the vaginal secretion, are the agents which destroy the 
bacteria. He stated further that the cervical canal, the uterus and the tubes 
are normally free from germs. 

In 1919 Giltner and Bandeen (20) compared the bacterial findings from 
ten puerperal uteri with those from the meconia of calves from the same 
uteri. They say, '" Thirty-four organisms were isolated from the A'aginae and 
twenty-four from the uteri. There were fifty-two different organisms isolated, 
only five of which occurred in both uterus and vagina. There were no two 
cows which showed an identical flora in either uterus or vagina, and there 
was very little similarity in the bacterial flora of any two cows, although they 
were all on the same premises ***** 

" Of the organisms found there Avere, of the four genera according to 
Migula, bacterium, 22: bacillus, 3: micrococcus. 11; streptococcus, 4." 

The bacteriology of the fetus and newborn infant is not uniform. It has 
not been carried out methodically in most cases and the investigator in human 



medicine has had access in the newborn infant only to tliose cavities which 
communicate with the exterior from which to make cultures. These cavities 
are the mouth, rectum and vagina. VonReuss (21) states that staphylococci 
are invariably present in the infant's mouth when they are present in the 
vagina of the mother. The streptococci are less frequently found. Kneise 
(22) reached the conclusion that 100% of infants are born with bacteria in 
the mouth and that Stajihylococcus aureus greatly predominates over the 
streptococcus. Knapp (23) reported finding a close relation between the 
bacterial flora of the vagina and intestine of the newborn. Schmidgall (24) 
found that in most cases the buccal cavity was free from bacteria for the first 
24 hours, although in a few cases she found hemolytic streptococci in the 
child's mouth as well as in the vaginal secretion of the mother during labor. 
She concluded that the vaginal flora in the newborn female depends upon the 
vaginal flora of the mother. The French investigators, Bonnaire, Keim, 
Jeannin and others, quoted by Mme. Brailowsky -Lounkevitch (25), reported 
that the buccal fluid of the newborn is sterile. Smith (26) found staphylo- 
cocci and streptococci in the vaginae of infants at a very early period after 
birth. The results of the work of Hymanson and Hertz (27) on the bacteri- 
ology of the partiirient vagina and the mouth and rectum of the newly boin 
are practically the same as those of the French workers. 

Various investigators have cultured the meconia of infants and have 
reported conflicting results. Some have found that the meconium is negative 
at birth, while others have isolated numerous bacteria. Very little detailed 
bacteriology has been carried out on the fetuses of domestic animals. The 
greatest amount of this work has been done on the bovine fetus and is com- 
paratively recent. When Bang (28) in 1896 flrst described the organism 
which is now called Bacterkmi abortum, he reported finding it in the tissues 
of the fetus. Besides a group of fetuses from which Smith (29) isolated 
Bacterium abortum, he mentioned three other groups, ''(a) the few fetuses 
yielding sterile cultures, (b) those yielding rapidly growing cultures of 
colon-like and other bacteria, and (c) those yielding pure cultures of a 
spirillum." Practically all of the work on bovine fetuses mentioned above 
has been carried out on fetuses expelled prematurely or dead. I have found 
almost no literature dealing with the bacteriology of the genital tracts of 
either the healthy or diseased fetus. 

Teohnic 

The technic which I have used in this work has been varied greatly during 
the four years. Practically every method of isolating microorganisms has 
been tried and very many different media have been experimented with. My 
main object has been to improve the technic, i. e., to make it less complex, to 
decrease errors from contaminating bacteria, and to find the best media upon 
which the largest number of organisms will grow. In any case many errors 
will arise and it is almost impossible to interpret results in precise terms. 

With the exception of the material obtained from the abattoir, all has come 
from herds located in six of the eastern states. The genital tracts from the 
cows and veal calves were removed in the following manner: After the 
animal was slaughtered and skinned, the posterior parts were elevated about 
four or five feet above the floor. An incision was made along the Unea alba. 
to allow the abdominal viscera to escape, after which the si/mphi/'Sis pubis was 
sawed through. An area around the genital tract was flamed with a gasoline 
torch. Then an incision was made through the recto-vaginal pouch and other 
folds of peritoneum between the rectum and vagina, four or five inches ante- 
rior to the vulva. A stout cord was passed through the incision around the 
vagina and tied securely. An assistant pulled centrally on the cord while all 
of the attachments of the genital tract were severed with a sterile knife and 
wrapped immediately in a sterile towel. In some cases, when an assistant 
was not at hand, the vulva was grasped with a pair of Bozeman's forceps, or 
other long forceps, held in the left hand, while the attachments were being 
severed. The only time that the genital organs came into contact with the 
hands or other possible infection carrier was when the cord was placed around 



the walls of the vagina and during the exposure for a very short time to the 
bacteria of the air before wrapping in the sterile towel. The tracts were 
then taken to the laboratory where cultures were made, smears of the 
different parts made on glass slides, and sections of the ditlerent organs taken 
for pathological study. 

Cultures were made from the ovaries, oviducts, right and left uterine apices, 
body of the uterus, cervix, and in most cases from the vaginal folds around 
the external os uteri. The mammary gland was cultured in many cases. 
About forty cultures were made from each adult genital tract. Four cultures 
were made from each part. At lirst two aerobic and twu anaerol)ic cultures 
were made, but later cultures grown at partial oxygen tension were made 
because no strict anaerobes were recovered. The ovarian cysts and cystic 
corpora lutea were cultured with a sterile, sharjj-pointed pipette which was 
pushed through the cyst wall after an area on the latter had been sterilized 
with a heated platinum spatula. As a rule the cultures from the cystic tluid 
were negative and_because of this Rosenow's (30) tissue crusher was used 
to crush tlie whole ovary or as large a section of it as could be placed in the 
crusher. Cultures were then made from the material that was ground up. 
This method is inconvenient for crushing cows' ovaries because the ovaries, 
especially those which are diseased, are too large as a rule to be placed in the 
crusher without sectioning and it is difficult to handle them so much with- 
out contamination. The simplest plan is to sterilize an area on the surface of 
the ovary with a heated platinum spatula, and with a tine pair of sterile 
forceps to tear out a small piece of the ovarian tissue about the size of a pea 
or smaller, and to place this on the medium. At times it is desirable to make 
cultures from the adhesions on the capsule of the ovary and this can be done 
in the same way without sterilizing tlie tissue, if the ovary has been handled 
under aseptic conditions. Generally if an organism can be isolated from the 
adhesion around an ovary, it can also be isolated from the ovarian tissue 
itself and it is not necessary to make cultures from both areas except for veri- 
fication. 

The oviducts were cultured by sterilizing an area with a heateil spatula 
and then making an incision into the lumen with a sterile platinum loop. 
Cultures were made through the fimbriated end of the tube if the latter was 
not adherent to the ovary. When the genital tracts of smaller animals were 
cultured, the lumen of the oviduct being too small for cultures, the whole 
ovary and oviduct on one side were crushed in Rosenow's tissue crusher or 
ground up with sterile sand in a mortar with a pestle. 

The apices and body of the uterus were cultured by tearing out a })ieee of 
the uterine wall, after sterilizing an area from which cultures were to be 
taken and making an incision through the wall with a sterile knife. If there 
were fluids in the uterus, they were withdrawn in sterile pipettes and placed 
on the medium. If the mucosa only was touched with a platinum loop and 
a bit of the glandular secretion placed on the medium, there was a failure 
as a rule to get a growth. The organisms, especially the streptococcus, 
appeared to be living in the depths of the tissue, as the submucosa, and not on 
the surface of the mucosa. We have demonstrated this, i. e., the streptococcus 
to be in the submucosa, in several preparations from sections and smears 
from the uterine wall, especially in chronic conditions. 

Cultures were taken from the cervical canal bv sterilizing an area on the 
ilorsal surface of the cer^ix, midway between the external and internal os 
uteri, with a heated spatula or a gas flame or torch. An incision was made 
with a sterile knife into the canal and a piece of the mucosa and submucosa 
was torn out with fine sterile forceps and placed on the medium. 

The anterior folds of the vagina and external os uteri were cultured in tlie 
same manner as described above. In calves and fetuses the mucus in the 
vagina was withdrawn with pipettes and cultured. The vulva and posterior 
part of the vagina were not cultured in all cases because much work has 
already been done on these parts and it was an impossibility to care for so 
many cultures from such a fertile field. Then. too. it was found that the 
bacterial flora of the posterior part of the vagina and vulva varied in diflFerent 
herds. 



AMien pregnant uteri were cultured, the same procedure was carried out 
with reference to the ovaries and oviducts. The fetal fluids were cultured 
and the fetal and maternal cotyledons, if these Avere large enough to secure 
pieces of the tissues. The apices and body of the uterus were cultured at 
the utero-chorionic space and tissues were also removed from the walls at 
these points. A piece of the cervical seal was removed, as described above, 
and cultured. Cultures were made from the anterior folds of the vagina as 
in the case of tlie non-pregnant uteri. 

For the most part the fetuses and calves were brouglit or shipped into the 
laboratory and post mortem examinations and cultures were made there. 
The alimentary tract was cultured at several different places by means of 
sterile pipettes, after sterilizing an area with a heated spatula. I'sually the 
rumen, abomasum, small intestine, caecum and rectum were cultured. The 
heart's blood, lungs, liver, spleen and genital tract were cultured also. The 
heart's blood was cultured l)y means of a pipette wliile the cultures from the 
other organs were made by placing a piece of tissue on the medium. 

When cultures were to be made from living calves immediately after l)irth, 
the latter were handled as follows: The cow was placed in a clean disinfected 
stall just liefore the calf was born. Her posterior parts were washed thor- 
oughly with a disinfectant and sometimes an entire Ijath was given. The calf 
was caught on a large piece of canvas whidi had been soaked for a long 
period in a strong disinfectant. Immediately after expulsion a sterile, soft 
rubber, horse catheter with a piece of glass tubing in which was a cotton plug, 
was inserted in the back of the calf's mouth and pushed down the esophagus 
to the stomach. (It is necessary to have an assistant witii aseptic hands 
hold open the calf's mouth.) Then the contents of the stomach were sucked 
up into the tube and tlie contents allowed to escape into a sterile container. 
The meconium can be collected in a similar manner. The anus of the calf 
should be disinfected, a sterile, soft rubber, horse catheter inserted into the 
rectum for a distance of 18 to 20 inches, and sterile water or saline solution 
from an irrigator run iiito the bowel and the meconium forced out. The 
meconium, wliicli is usually covered with a tough mucous coat, should be 
collected in large sterile Petri dishes or other containers and cultured. The 
'surface of the meconial mass can be sterilized with a heated platinum spatula 
and material from the center of the mass placed on the medium. 

Two or three smears were made upon glass slides from nljarly every tissue 
from which a culture was made. The smears were stained with methylene 
blue for various lengths of time, from five minutes to forty-eight hours. 
Duplicates also were stained with Gram's stain and sometimes carbolfuchsin 
was used. This was done to check up the cultures in case dead bacteria should 
be present or living organisms which were not being recovered in culture. 

Samples of blood were drawn from nearly all animals and the serinu set 
M-ith an antigen made from Bacterium ahortnw. Each genital tract of the 
fetuses and calves was carefully described at tlie time when cultures were 
made. 

Smillie's (31) method of injecting Guinea pigs and isolating Bacterium 
ahortum was em])loyed quite generally with the different tissues from which 
cultures were made. As a rule extracts from ovaries and oviducts were not 
injected but contents of the uteri, extracts from uterine walls, cervical seals, 
vaginal secretions and fetal fluids, extracts from fetal membranes, and c<m- 
tents of the alimentary tracts of fetuses, as well as extracts from other 
tissues, were injected. One extract AA'as made from the lung, liver and spleen 
of a fetus and another from the ijenital tract. These extracts were groiind 
iin in sterile mortars with sterile sand and a sterile saline solution, then 
filtered through cotton. One or two c.c. were injected subcutaneously accord- 
ing to the size of the pig and the dilution of the extract. In cases where 
it was possible to olitain samples of milk, they we're centrifuged down and 
+lie sediment injected into the pigs. In three or four Aveeks the pigs were 
slaughtered by placing them in jars filled with artificial illuminatin£r gas. 
Each pig. with its abdomen dorsal, was than stretched on a board * and 



* The construction of this Iward was suggested to me by Dr. H. Hoffman. 



fastened to it by means of four nail points arranged at angles at each corner 
of the board, each nail piercing a leg of the pig. The board (See Plate 1) is 
easy to construct and saves much time when large numbers of guinea pigs are 
to be examined, for it does away with the tying of strings or fastening of 
clamps as is the case with most animal holders. By this method we have 
been able to slaughter and culture about twelve jugs in an hour. 

The hair on the abdomen was singed ofl" with a gas flame, tbe charred liair 
scraped off, and the surface swabbed with ;■>% solution of phenol. An incision 
was made on the median line and the right thoracic and abdominal walls 
cut away. This allowed the intestinal tract to be removed easily, as shown 
in Plate II. Then with sterile forceps and a knife, the spleen, liver, and 
uterus or testes were removed and placed in sterile Petri dishes. Cultures 
were made from these organs by placing a piece of tissue upon the medium. 
The cultures were sealed with wax, as Smith suggests, incubated and treated 
according to the last method described under the different methods of pro- 
ducing jjartial oxygen tension. A sample of lilood was taken from each pig 
and set with an antigen made from Bacterium abortuni. 

When the work was first begim, aerobic and anaerobic cultures were made 
from the different tissues. Liborius' (.32) method for making the anaerobic 
cultures was used. Cultures grown under partial oxygen tension were made 
and altogether four different methods were tested. First. Nowak's (33) 
method of placing cultures in an atmosphere where B. siibtilis was growing; 
second, Wright's (34) method with pyrogalic acid and 0.1% solution KOH 
(one part liquor kali, caust. to ten part H.O). 

A third method is the following: Culture tubes are placed in large jars 
with ground glass stoppers. An alcohol lamp is set in the jar and lighted, 
vaseline is smeared over the ground surface of the cover and the cover placed 
on the jar. It is necessary to have a hole, 3 to 4 mm. in diameter, bored in 
the cover. Before placing the latter in position, the hole can be filled with 
sealing Max. The flame burns only until the oxygen has been consumed and 
a vacuum has been created. When the cultiires are to be examined, great 
difficulty will be experienced in removing the cover unless the sealing wax is 
removed first to allow air from the outside to rush in and replace the vacuum. 

The fourth method is as follows: The cotton stoppers of the culture tubes 
may be sealed either with paraffin or sealing wax. In sealing tubes with wax, 
care should be taken not to push the plug below the mouth of the tube because 
otherwise it is hard to remove the wax without breaking the tube when the 
latter is opened for examination. The plan is to form a cap over the cotton 
plug without allowing any wax whatsoever to drop inside the mouth of the 
tube. Before sealing the tube, the stopper should be trimmed and flamed. 
This allows the bacteria which have been placed on the medium, access only 
to the oxygen in the air sealed up in the tube. 

The best results have been obtained from sealing the tubes with wax or 
paraffin. There is much less labor involved when paraffin is used but it means 
very dirty, disagreeable work when the cultures are examined. Nowak's and 
Wright's methods, as well as tlie covered jar with the alcohol lamp, are some- 
what complicated and cumbersome. It was found that they produced a good 
partial oxygen tension for growing Bacterium abortum but so much condensa- 
tion water formed in the jars that the growth of streptococci was inhibited to 
a certain extent. 

ISIany different media have been used throughout the work. For a long 
time duplicate cultures Avcre made on various media and grown under diverse 
methods, until we were certain whicli medium and which method was the 
best. Cultures were made on ordinary nutrient agar, solidified bovine serum, 
Loeffler's (3o) blood serum, glucose glycerine agar plus sterile bovine serum. 
Huntoon's (36) hormone agar, splenic extract bo\iillon, ovarian extract 
bouillon, and uterine extract bouillon. All these were prepared in the same 
manner as ordinary peptonized bouillon. Agar was made also, according to 
Himtoon's method, from the spleen, ovary and litems as well as from genital 
tracts in early stages of pregnancy, the fetal fluids and membranes bein? 
ground up with the other tissues. Ordinary nutrient veal agar, or nutrient 
agar made from peptonized beef bouillon, upon which has been placed either 



a little sterile serum or a few sterile blood corpuscles collected from a horse, 
cow or calf, has been employed extensively and with very good results. The 
Hydrogen-ion concentration of the culture medium seemed to play an import- 
ant part, especially in the cultivation of the streptococcus. At first a medium 
was used with Hydrogen-ion concentration of pH. 6.5 but it was found that 
media at pH. 7 or 7:5 was much more satisfactory. 

In many instances we did not use the blood corpuscles or serum but. instead, 
a piece of tissue, from which the culture was made, was placed on the 
medium. The object is to have some fresh enriching jnaterial to favor the 
start and gro\vth of the organism transplanted with the tissue. This combi- 
nation, i. e., nutrient agar (pH. 7-7.5) with some enriching substance such 
as serum, blood corpiiscles, or a piece of fresh tissue, and cotton stoppers 
.<;ealed with sealing wax, has given very favorable results and has yielded 
growths when other media have failed to do so. 

All cultures were incubated at 37 degrees C. Examination was made 24 
to 48 hours after incubation and if no growths were observed, the wax caps 
were removed and the tissue or other material which had been introduced 
into the medium was smeared over the surface of the latter and pushed down 
again into the condensation water. The stoppers were resealed and the tubes 
placed back in the incubator. If in 48 hours more no growth appeared, the 
same process was repeated. It is surprising to find how many more growths 
can be obtained by this procedure. This is especially true in the case of the 
streptococcus and Bacterium ahortum. If no growth appeared after 10 or 12 
days, the cultures were discarded. 

Bacteriological Findings 

In order to simplify the work, all the animals whose genital tracts have 
been cultured have been divided into six groups. Group I consists of genital 
tracts from pregnant cows. Group II comprises genital tracts from non- 
pregnant uteri. Group III is virgin heifers. Group IV is made up of calves 
from 3 to 12 Aveeks of age; Group V consists of calves 1 to 3 weeks old; 
and Group VI of aborted fetuses or those removed from pregnant uteri. 
More work has been done on Group II than on the other groups because of 
the greater ease with which the non-gravid tract is obtained. The majority 
of these have been cows with abnormal breeding histories. An abundance of 
material could be had from local slaughter houses and larger abattoirs but 
one is handicapped witli such material because he knows nothing of the history 
of the animals. The history has been considered an important factor, especi- 
ally in connection with the work on the non-pregnant genital tracts. Many 
tracts in which conception had not occurred have been observed, without 
apparent lesions, at least nothing that is considered pathological at the pres- 
ent time could be found. Other factors which might have caused failure to 
breed, such as a diseased male, had been eliminated. Therefore, in considering 
a normal genital tract for bacteriological examination, the breeding history 
seemed very important but we found that it was difficult to obtain such 
information. 

The genital tracts of 139 females have been examined and in nearly every 
case each organ of the tract was cultured. The following divisions were 
made: Genital tracts from pregnant animals. 11; normal genital tracts from 
non-pregnant animals, 19; abnormal genital tracts from non-pregnant 
animals, 50; genital tracts from virgin heifers, 10; calves 3-12 weeks old. 
12; calves 1-3 weeks old. 9; aborted female fetuses, 13i: aborted female 
fetuses whose dams were injected with Bactermm ahortum, 2 : normal female 
fetuses, 11 ; fermile fetuses removed from cows suffering from dystocia. 2. 
Twenty-four male fetuses, normal, aborted, and dystocia cases were examined. 
Besides culturing the genital tracts of the fetuses and calves 1-3 weeks old, 
a bacteriological examination was made of the tissiies from the rest of the 
animals. 

The results of this bacteriological investigation have been extremely inter- 
esting. The exact locations of organisms isolated from different parts of the 
genital canal may be seen on the charts. In manv cases all of the cultural 



.8 

characteristics of an organisia have not been worked out farther than identi- 
fying it according to Migula's classification. It is often impossible to obtain 
a subculture of an organism from the primary growth. A complete study of 
the characteristics of certain of these organisms will be carried out in detail 
and published later. 

The cultures from the genital tracts of the 11 pregnant animals (Group I — - 
Chart I) were uniformly negative in five cases. Bacterium abortiitn was 
isolated from the fetal fluids of one animal while the other cultures from this 
tract were negative with the exception of the vagina where B. coli was found. 
Number 6 was negative with the exception of the right uterine apex in which 
there w^as an unidentified rod, and the vagina in which were B. coli and Sta- 
pltylococcus albits. Tract 8 gave pure cultures of a streptococcus from nearly 
every organ, while in Tract 2 growths were obtained only from the uterus 
and cervix. In the uterus of Tract 3 streptococci mixed with Ps. pijocyaneus 
were found in the uterus; the external os uteri yielded only a streptococcus. 
The ovaries and right oviduct of Tract 11 gave cultures of Streptococcus viri- 
dans in conjunction with staphylococci. From the anterior folds of the vagina 
of this tract also was obtained a culture «f Staph iilococcus albus. 

Group II (Charts II, III), in which I have included 69 non-pregnant 
animals, is of unusual interest. Eighteen are reckoned as normal tracts and 
fifty-one are either pathological or came from cows with abnormal breeding 
histories. Of the 18 normal genital tracts, 5 yielded no growths, while 6 gave 
cultures from the uterus only, the remainder of the tracts being negative. 
Four tracts showed cultures of a streptococcus from some part of the uterus 
and also from one of the oviducts. In one case the streptococcus was mixed 
with a Staphylococms albus. Another tract yielded growths of a strepto- 
coccus and Staphylococcus albus from the ovaries and oviducts and Ps. pyo- 
cyaneus from the uterus. Still another tract gave cultures of Stapliylococcus 
albus from the oviduct and a Gram negative imidentified rod from the cervix. 
Tract 54 was negative wath the exception of a Staph jjlococcus aureus from the 
cervix. 

Under Group II are also included 17 animals slaughtered in one herd. 
These cows varied greatly in age, 4 being heifers that had never conceived 
though bred many times. All had been douched regularly and otherwise 
treated for sterility for many months. Cultures were obtained from some 
portion of each tract. All of the organisms isolated from these animals fell 
in the group of streptococci, with the exception of B. coli which was fovmd 
once in the anterior part of a vagina. The majority were streptococci coming 
chiefly from the oviducts and ovaries. Occasionally a pure culture of a 
streptococcus was recovered from the anterior portion of the vagina, the cervix 
or the uterine apices. In a few cases a staphylococcus was recovered in pure 
culture from the various organs of the genital canal but usuallv a strepto- 
coccus was found with it. A saccharomyces was isolated from the uterus of 
one case. Number 48. 

The remaining 34 genital tracts from Group II Avere collected from 17 
various herds which in most cases Avere long distances apart, 100 to 300 miles 
or more. With the exception of Tracts 36, 66 and 67, which were obtained 
from the abattoir and the history of which was not known, and of two cases 
of pyometra, all of these cows were slaughtered after being condemned for 
incurable sterilitv. All had received various kinds of treatment for over- 
coming this condition. For the most part the bacteriological findings were 
similar to those of the above mentioned 17 cows. The predominating organ- 
isms were cocci, chief among them being the streptococcus. Staphylococci and 
occasionally a sarcina were recovered but in nearly every case these were 
found in coniunction with the streptococcus. Onlv three tracts (8', 9, 61) 
from this series gave negative results and in only one were growths confined 
to the uterus alone. In the oviduct of one cow (39) a paracolon organism 
was found and in a case of hydrosalpinx (2), an organism from the colon- 
aerogenes group was isolated in pure culture. Bacillus pifogenes and Barillns 
aerogenes capsulatus were recovered respectively from two cases of pvometra 
(21 and 24) but in each case a streptococcus was found with the orsranisms 
named. Bacterium abort um was found in the udder of one heifer (4) that 



9 

had never conceived. A hemolytic streptoeoceus and Bacterium ahortum were 
isolated from a tract (30) which was affected with granular venereal disease. 
Occasionally spore-bearing rods have been recovered from the cervix and 
several times from other parts of the genital canal. In one or two cases 
other unidentified rods have been recovered in cultures. A fungus also has 
been isolated in one or two instances. 

Groui> III (Chart IV), which is comprised of virgin heifers, showed fewer 
bacteria than the groups of pregnant or non-pregnant animals. Six of the 
ten heifers failed to show any growths, while one (7) yielded a spore-bearing 
rod from the cervix. Number 2 showed a strepto(^occus in both tubes and 
apices of the uterus, while 8 showed two strains of streptococci. Streptococcus 
inrdans and Streptococcus hemolyticus (Beta), in the uterus, and a growth 
of Streptococcus viridans in the external os uteri. Xumber 10 showed 
kitreptococcus viridans in both ovaries and in the body of the uterus. 

Group IV (Chart V) consists of 12 calves from 3 to 12 weeks of age. The 
udders of calves and fetuses were cultured more frequently than were those 
of adult animals. There was practically no glandular tissue present but, 
after cutting off the teats, pieces of the fat and connective tissue were 
removed with sterile forceps and placed on the media. The genital canals of 
eight of these animals yielded no growths of microorganisms although Bacte- 
rium ahortum was recovered from the udder of Number 9, which is included 
in this group. In three cases (3, 4, 10) streptococci were isolated from the 
ovaries. In two instances (3, 10) they were associated with Staphylococcus 
aureus. Number 4 gave growths of Staphylococcus aureus, albus and Strepto- 
coccus viridans from the vagina, while the vagina of number 10 gave a pure 
culture of B. coli. From the uterus of Number 10 was isolated Bacterium 
ahortum. The tract of Number 2 was negative with the exception of Strepto- 
coccus viridans in the vagina. 

Group V (Chart VI) is made up of genital tracts removed from calves 
1 to 3 weeks old dying from various types of calf infections, such as pneu- 
monia, enteritis, etc. Six of these tracts failed to show any bacteria. Case 6 
gave pure cultures of Staphylococcus alhus from the uterus, the rest of the 
tract being negative. 

Group VI (Charts VII, VIII, IX, X, XI), in which I have included 62 
fetuses ranging in age from 30 days to 9 months, is comprised of 23 fetuses 
removed from pregnant uteri, 25 aborted fetuses, 5 aborted fetuses whose 
dams were injected intravenously with Bacterium ahortum, 6 fetuses that 
were removed from cows suffering from dystocia, and 3 mununified fetuses. 
The charts upon which the bacteriological data for this group are given 
show from what tissues, besides the genital tract, microorganisms were 
isolated. 

In the case of the 23 fetuses removed from pregnant uteri, the majority 
appeared normal, although the exact result of the pregnancy, had the fetus 
remained in utero, cannot be known. Bacterium ahortum was recovered from 
three of these fetuses. In one of these. Number 26, Streptococcus viridans 
also was isolated from the meconivim in the rectum, while Bacterium ahortum 
was not found there. Cultures of streptococci were obtained from 8 fetuses, 
although in five cases they were found in conjunction with Staphylococcus 
aureus or alhus. Four fetuses yielded growths of staphylococci, while 8 failed 
to show growths of any organism. 

Bacterium ahortum was recovered from 10 of the 25 aborted fetuses (Chart 
VIII). In 4 cases Staphylococcus alhus or aureus was recovered. Strepto- 
cocci were isolated from 6 fetuses, 4 cases of these yielding no other organism, 
while in two cases, 15 and IG, organisms from the colon-aerogenes group v/ere 
recovered. The stomach contents of Fetus 15 contained a fungus also but 
this fetus was expelled alive and had swallowed much extraneous material. 
A Gram positive spore-bearing rod was recovered from the liver of Fetus 2. 
Staphylococci and organisms from the colon-aerogenes group were recovered 
from Fetus 4, which was expelled alive. Fetus 33 yielded pure cultures of 
B. prodigiosus from the heart's blood and small intestine. A hemolytic 
Staphylococcus albus was isolated in pure culture from Fetus 34. From the 



10 

stomach contents and intestinal tract of Fetus 54 was recovered a very inter- 
esting fungus, and Staphylococcus albus was found also. A Gram positive 
organism similar to B. pyogenes and from which I was unable to get a growth, 
was observed in smears from the viscera of Fetus 58, which was only IVo 
months old. The uterine exudate from the dam gave a pure culture of 
B. pyogenes and the organism in the fetus was apparently the same. Fetus 
61 gave a pure culture of a short Gram positive rod, which has not been 
identified, from the stomach contents. Only two fetuses in this group failed 
to give growths of any organism. 

On Chart IX are tabulated the bacteriological findings of 5 fetuses whose 
dams were injected intravenously with Baoterivm abortiim. This organism 
was recovered from some part of each fetus but was found in pure culture in 
only one case. In the other four cases staphylococci also were recovered and 
in Fetus 32 Streptococcus viridans was found. 

Two of the six fetuses which were removed from cows suffering from 
dystocia (Chart X) failed to show any bacteria in cultures. Fetus 5 gave 
a growth of Streptococcus viridans and a Staphylococcus alhus (hemolytic). 
From all the tissues of Fetus 31 a fungus was recovered. Fetuses 55 and 56 
were twins and from the tissues of both of these an organism that was either 
B. paratyphoid B or B. enteriditis was recovered. Fetus 31 showed extensive 
emphysema. The others failed to show any signs of this condition. 

Chart XI gives the bacteriological data from three mummified fetuses. Fetus 
20 gave cultures of Streptococcus viridatis and from the spleen an unidentified 
Gram negative rod was recovered. An organism from the colon-aerogenes 
group was recovered from Fetus 49. while Fetus 51 showed the same organ- 
ism in pure culture. Guinea pigs inoculated with extracts of the tissues from 
this fetus, however, showed typical lesions of Bacterium abortum and it was 
iecovered in pure culture from their spleens and livers. 

The bacteriology of the genital tracts of the fetuses has been of unusual 
interest. As a rule the distribution of the bacteria throughout the genital 
canal of the fetus cannot be traced as well as in the adult because of smaller 
size of the former. The cultures that were isolated directly on the media 
came for the most part from the uterine cavity, although in some cases 
Bacterium abortum was not recovered in culture but from guinea pigs that 
had been injected with extracts made from the genital tract. No cultures 
were isolated from the vaginae of the fetuses. In no case were bacteria 
recovered from the male or female genital tracts of fetuses removed from 
pregnant uteri. The genital organs of 8 female and 7 male aborted fetuses 
were negative. Bacterium abortmn was recovered from the reproductive 
organs of 3 females and 2 males, the organism being found in conjunction 
wnth Staphylococcus aureus in the case of one female. Tlie genitalia of 2 
males whose dams were injected with Bacterium abortum were negative, 
while the genital tract of a third male yielded Bacterium abortum and 
Streptococcus viridans. From the genital tracts of two females in this 
group Bacterium abortum was recovered but in the case of one of them, Fetus 
39, Streptococcus viridans, Staphylococcus albus and Staphylococcus aureus 
were also recovered. 

The results were negative from the genital tracts of one female and two 
male fetuses that were removed from cows suffering from dystocia. A fungus 
was recovered from the testes of one male. This fungus was also isolated 
from the other tissues of the fetus. The genital organs from the pair of 
twins. Fetuses 55 and 56, a male and female in this group, gave pure cultures 
of B. paratyphoid. B or B. enteriditis but, as mentioned before, the same 
organism was also recovered from the other organs of these fetuses. The 
bacteriology of the genital tracts of the miunmified fetuses could not be 
worked out satisfactorily. 

The different microorganisms isolated have in a general way been constant. 
Tlie majority of the cultures have fallen into the group of cocci. Streptococci 
have predominated but staphylococci have been quite common. The strepto- 
cocci most often found associated with the genital organs of cattle and fetuses 
are those which belong to the rnridans group. The majority of these have 
been shortchained and composed of rather large, distinctly oval segments. 



11 

In many eases only pairs were observed hut this was probably due to the 
medium and to tlie length of time the culture was grown after isolation. 
Quite a large number of hemolytic streptococci belonging to the Beta type 
have been recovered, as well as a few strains belonging to the group that 
Brown ( 37 ) calls " alpha prime." In the latter case immediately surround- 
ing the colony is a narrow zone of corpuscles that have not become hemolyzed, 
while surrounding this zone is a broader one in which hemolysis of the cor- 
puscles has taken place. The various results of the inoculations of these 
streptococci from the genital canal made upon the sugar media have not 
yielded anything definite as yet. There seem to be many slight variations in 
each strain which make anything like classification at present difficult. 

The staphylococci have been mainly ^staphylococcus alhns and Staphylo- 
coccus aureus. Some of them have been hemolytic and some non-hemolytic. 
A larger percentage of the cultures of Staphylococcus aureus has been hemo- 
lytic than of Staphylococcus albns. 

Iiu.cteriuni abortuui was recovered from many of the different genital 
organs. Before definitely designating an organism Bacterium ahortutn, I 
have injected it into guinea pigs to see if it produced the characteristic 
lesions, and have made antigens from the cultures and checked them against 
positive abortion serum. The other biological and staining characteristics 
have also been carefully worked out. 

A few Gram positive spore-bearing rods and Gram negative rods which 
could not be identified were isolated at various times, especially from the 
cervi.x and anterior part of the vagina, as recorded on the charts. Occasion- 
ally organisms from the colon-aerogenes group, paracolon types, Ps. pyocya- 
veus and B. pyogenes liave been recovered but their ciiltural characters are 
too well known to need description. 

Experimental 

In connection with this investigation, experimental work has been carried 
on with calves of various ages and with a limited number of heifers. The 
purpose of these experiments has been to find : First, what the effect of some 
of these organisms would be upon the reproductive organs of young animals 
if bacteria were injected directly into the uterine cavity ; second, whether the 
genital canal could free itself of infection when bacteria were injected 
directly into the uterine cavity, and, if it could do this, how long it would 
take for the microorganisms to disappear ; third, whether these bacteria 
had any affinity for, or would localize in, the genital canal, if suspensions of 
the organisms were injected intravenously. 

In case of the males the different suspensions of bacteria were injected into 
the scrotal sac or testicular tissue with a hypodermic syringe. The uteri 
of the females were exposed through the flank after laparotomy had been per- 
formed, and the bacterial suspensions injected with a hypodermic syringe 
directly into the uterine cavity. As a rule the udders of the calves were 
injected with the same organism which was placed in the uteri. Intrave- 
nous injections were made in the usual manner. 

Very heavy bacterial suspensions of Bacterium abortum were used, about 
twice as cloudy as a bouillon culture of }{. tj/phosus. Four different strains of 
the Bang organism were used, two from the U. S. Bureau of Animal Industry 
and two isolated from aborted fetuses in our own laboratory. The cultures 
were grown on nutrient agar slants, washed off with sterile saline solution, 
and mixed before injection. The streptococci which were used were isolated 
from the genital tracts of sterile cows showing extensive lesions, such as 
adhesions of the different parts. These saline suspensions were not so hea\y 
as those made from Bacterium abortum. B. proteus vulgaris, which had been 
isolated from a case of pyometra in a bitch, was injected into the udder and 
uterine cavity of Experimental Calf 15. 

After various periods of time, the animals were slaughtered and the genital 
tracts of the females were removed and ciiltured as described under technic. 
The testes, epididymes and seminal vesicles of the males were cultured after 
removal in the following manner. A string was tied tightly around the neck 



12 

of the scrotum and tlie latter was severed between the string and the abdomi- 
nal wall before tlie abdominal cavity was ojKjned. The testes and epididymes 
were lenioved after an incision was made in the abdominal wall on tlie median 
line and the symphysis pubis severed. Tlie mass of fat at the neck of the blad- 
der which usually surrounds the seminal vesicle was cut away with sterile for- 
ceps and knives and the vesicles removed with it. These were placed in 
sterile towels, taken to the laboratory and cultured in the same manner us 
similar tissues. 

Thirty-three animals were used in this exj)crimental work (See Chart X"ll). 
12 males and 21 females. They were kept in isolated stalls with tight parti- 
tions, clean straw being used for bedding. Most of the calves had nothing but 
whole milk fed to them frcmi individual pails. One or two older animals had 
some hay and grain. 

The scrotal sacs of 3 males were injected with 1 c.c. of a living culture of 
Bacterium uhorluin and two with a live suspension of a Streptwocciifi viri- 
dans. These were slaughtered in from 17 to 1?> days. A streptococcus was 
the only organism recovered and this was found only in an atrophied testicle 
of one of the calves. Six more males were injected intravenously witl: 
10 c.e. of the Bacterkitn abortum suspension, many of the animals receiving 
the injection twelve times, making a total amount of 120 c.c. In none of 
these cases was Bacterium abortum recovered from the testes, epididymes 
or seminal vesicles. In one case (23) a 10 c.c. injection of a streptococcus 
was made following an injection of the Bang bacillus per vein but the cul- 
tures from this tract were also negative. These six animals were slaughtered 
in from 3 to 4cS days after the first injection. 

One female calf received an injection of 1 c.c. of a suspension of Bacteriuvi 
abortum in her udder. She was slaughtered 51 days after injection but cul- 
tures from her genital tract and udder were negative with the excejition of 
one colony of Htaphylococcus albus frcmi one ovary. The uteri and udders of 
four other females were injected with five c.c. and 3 c.c. respectively of a 
suspension of Bacterium abortum,. The abortion organism Avas recovered from 
the uterus and vagina of Calf 7 of this group, after having been inoculated 
15 days, and also from the uterus of Calf 13 which was slaughtered after 
having been inoculated 65 day^-. The organism was not recovered from the 
other two females although an organism from tlie colon-aerogeues group was 
recovered from the uterus and udder of Experimental Calf 14 and Ps. pj/o- 
ci/aneus from her vaginal muciis. The udders of all these females were nega- 
tive. The uterus of Experimental Calf 8 Avas injected with a culture of 
i^trcptococeus viridans is(jlated from Fetus 32. Twenty-five days later the 
organism was not recovered. Experimental Calf 10 received 5 c.c. in the 
uterus and 3' c.e. in the udder, of a culture of streptococcus isolated from 
the genital tract of a sterile cow that had endometritis, salpingitis an<l 
ovaritis. Sixteen days later the calf was in excellent health and was 
slaughtered for veal. An examination of the genital tract showed that the 
uterine apices, ovaries and oviducts were all adherent in a mass of fat. 
The pavilions were adherent to the ovaries, in fact the lesions were very 
similar to those that have been found in the sterile animals which we have 
examined. A streptococcus with identical characteristics of the organism 
injected was recovered in pure culture from tlie adhesions and frona the 
mucus in the vagina. 

Two other females, 31 and 32, were injected intravenously with cultures 
from Streptococcus viridans isolated from the genital tract of a cow suffering 
from hydrosalpinx and cystic ovaritis. Each received three injections, one 
of 5 c.c. and two of 10 c c, of the suspension over a period of eight days. 
Calf 32 developed a severe arthritis in the left stifle joint from which she 
did not recover. Tlie calves were slaughtered in 73 davs after the first 
injection but there was no opportunity to culture the affected joint. The 
cultures from the genital tracts were negative. 

Five females were iniected intravenously with 10 c.c. of Bacterium ahortum, 
several receiving this injection 13 different times. Bacterium abortum was 
not recovered from the genital tract or udder of any of this group of animals. 



1 "^ 

One small female calf (30), expelled preniatuiely ait eight nionths, was 
injected intravenously with 10 c.c. of a suspension of Bacterium abortum. 
The calf died the following day and Bacterium, abartum was isolated from 
her uterus. The cultures from the heart's blood and spleen were negative. 
The dam of this calf had retained jjlacenta but Bacterium abortum was 
not recovered from it, although her serum agglutinated an antigen made 
from the organisjn. 

The uterus and udder of Experimental Calf 15 were injected with a cul- 
ture of B. protcus vuUjaris, 4 c.c. in the uterus and 2 c.c. in the udder. 
The organism had been recovered from the uterus of a liitch suffering from 
pyometra. The calf was slaughtered 37 days later and the organism not 
recovered, but a staphylococcus was isolated from the uterus. 

Two female calves were kept as checks under the same conditions as the 
calves that were injected. The cultures from their genital tracts were nega- 
tive. Three animals are still living, experimental Calf 11, aged Hi months, 
12. aged 17 months, and 18, aged 6 months. Observations are being con- 
tinued on these animals. The uterus of number 11 was injected with 5 c.c. 
of a culture of streptococcus viridans isolated from the genital tract of a 
sterile cow and the same amount was injected into her udder. At present 
she is in good condition and comes in estrimi regularly. Number 12 received 
10 c.c, of a live suspension of Bacterimn abortum in her uterine cavity and 
,5 c.c. of the same suspension in her udder. She is now pregnant 4'/. 
months and in good condition. Calf IS received intravenously 10 
c,c, of a live culture of Bacterium abortum five times. The injections began 
when she was 20 days old ajid were c(jntinued over a period of one month, 
She is at the ])rcsent time apparently in excellent health. 

Boctcrium, abortum was not recovered from the genital tracts of the male 
or female calves that received injections of this organism intravenously, 
with the exception of Calf 30 as mentioned. It was recovered from the 
spleens of all the calves with the exception of Calf 23 whicli died the day 
following the injection, and Calf 24 which was slaughtered the day after 
ijijection. In one case Bacterimn (ihortuw v.as isolated from the mediastinal 
lym|)h glands and also from the two lymph glands removed from the pelvic 
cavity. Undoubtedly it would have been recovered from lymph glands in 
other cases had cultures been taken. In several cases Bang's bacillus was 
recovered from the lungs also. 

The spleens of many of the calves from which Bacterium abortum was 
reroverei were enlarged, in one or two cases being twice as large as normal. 
The splenic pul]i was increased in amount and much darker in color than 
it should have l>een and the corpuscles enlarged. In other cases the spleen 
was very friable. These conditions are very similar to those observed in the 
spleens of guinea pigs a short time after they have been injected (10 to 14 
days). Apparently this organism has an affinity for the same tissues in 
the calf as in the guinea pig. Although abscessation of the tissues, such 
as the liver, epididymes, lungs, etc.. was not ]uodured. it might be done if 
proportionately the same amount of injected material should be given the 
calf as the guinea pig. The atrophying of the testicle, as seen in guinea 
pigs, was not observed in any of the calves injected with Bacterium ahortum. 

Although in cases where Bacterium ahortnm or the other organism injected 
M"as not isolated from the genital organs at time of slaughter, staphylococci, 
streptococci and other miscellaneous organisms were sometimes recovered, 
Intra-uterine and intravenous injections seemed to have a detrimental effect 
upon a majority of the calves, especially the youngest animals. In many 
cases they showed a temporary diarrhea or came down with pneumonia. The 
grnitnl tracts from those animals which did not thrive during the experi- 
n\ents were the ones from which bacteria were recovered a< a rule. 

The serum of all the animals except those that died or were killed a 
verv short time after injection (23, 24, 30), aaglutinated a Bacterium abortum 
antigen in dilutions of 1 to 600. This was the highest dilution made. The 
method of iniection, intravenous, intra-uterine. udder or scrotal sac, seemed 
to make no difference in the reaction. 



14 

UlSCUSSIOX AND SUMMAKY 

To judge from the above work it seems that there is a similarity between 
tlic bacteriology of the human female genital tract and that of the female 
of the bovine species. In the human female there is found the gonococcus, 
while in cattle Bacterium abort mn has a])'parently adapted itself peculiarly 
Ut the pregnant animal. Aside from these difi'erences the bacterial flora 
of the reproductive organs of the two species is very much alike. The 
streptococcus is found predominating in cattle and has been isolated from 
174 difl'erent parts of all the genital tracts examined, the staphylococcus 90 
times, Bacterium abort urn 17 times, organisms from the colon-aerogenes 
grouj) 4 times, Bacillus pyogenes capsulatus ;i times. Bacillus pyogenes 
3 times, sarcina 3 times, I's. pyocijaneas 3 times, fungi 3 times, B. para- 
tfjoJioid B twice, paracolon once, and unideiitilied rods eight times. 

From these data it apj)ears that the genital tracts of cattle should nor- 
mally be free from bacteria. The results obtained from the younger aninuils 
seem to support this conclusion although microorganisms are sometimes 
foiuid in their genital organs. There seems to be a close relationship between 
Ihe presence of bacteria iii the genital canals of calves and the various 
symptoms of calf infections observed, especially enteritis and pncinuonia. 
When a calf has sulTered from such infections, especially from enteritis, and 
has recoveied, one can, as a rule, isolate, bacteria from the genital canal 
of thiit calf if it is slaughtered soon after recovery is made. The type 
of infection may vary and the amount will vary according to the severity 
of the disease. If the animal is slaughtered even several months after 
recovery, there is also a greater possibility of recovering microorganisms 
in culture from the genital canal than from the tract of a calf or heifer 
that has not suffered from disease. 

The genital tract of a fetiis or young calf is almost c(mipletely filled v.ith 
very viscid, tough, faintly cloudy nuicus. The vagina is usually greatly 
distended with this fluid and remains so for the first four or five weeks 
after birth. After this time the amount seems to decrease and only the 
walls of the vagina are covered with it, although after puberty, when the 
animal comes in estruni, a large amount of this mucus is secreted. Undoubt- 
edly this has no other than a mechanical action for cleansing the vagina. 
The nuicus of estnnn differs from the mucus found in the genital canal at 
birth, the former being very clear and transparent. The latter has been 
collected with sterile pipettes from the vaginae of calves, placed in vitro, 
and tested for sterility. Streptococci and other organisms have been placed 
in the nnu-us and left for 48 hours after which time cultures were again 
made. 1 have not failed to recover the organism which I had placed in the 
mucus. FTirther, if the mucus and organisms were incubated for 24 hours, 
heavy growths of the organisms were found in many cases wiien smears 
from the mucus were made. The reaction of the mucus in the genital canal 
of the fetus, when tested wnth litmus paper, is distinctly alkaline or neutral. 
It cannot have, therefore, a very marked bactericidal action. 

Observations have been made as regards the matting of the hairs of the 
vulvar tiifts of these calves, all the animals having been kept imder the 
same conditions. It has been found that when the hairs are clean and free 
from fecal matter, pus aTid mucus, cultures from the mucus in the anterior 
part of the vagina are practically always negative, while cultures made 
from the vaginae of calves with vulvar hairs soiled with excretions and 
secretions, practically always yield bacteria. This factor seems to be a 
safe criterion for differentiating a vagina free from bacteria from one con- 
taining microorganisms in young females, and no doubt bears some relation 
to the bacterial content of the reproductive organs of adult animals. In 
the immature animal it has been found that if the anterior part of the 
vagina is infected with different microorganisms, the uterus also will con- 
tain bacteria. 

The genital tract of an adult cow with a normal breeding history is compar- 
atively free from bacteria, especially the ovaries and oviducts, cervix and 
external os. In the case of distinctly diseased genital organs, the majority 



15 

cf the microorganisms were isolated from the uterine adnexa. Approximately 
the same number of bacteria were recovered from the diseased uterus as from 
the uterus of the normal individual. As a rule, the uteri of the diseased 
tracts had been douchexl with various antiseptic solutions or treated in other 
ways, and this possibly had an influence upon the bacteria in this organ, 
keeping the number as low as or even lower than in normal cases (See 
graphs). Generally only a few colonies, or possibly a meagre growth, 
have been noted on most of the cultures. This is especially true of the 
streptococcus which has been recovered so many times, and may be due to 
the artificial medium upon which the bacteria are gro^Ti. 

Since the majority ol the cultures from the genital tracts of the pregnant 
uteri, as well as most of the cultures from the fetuses removed from the 
latter, were negative, it is difficult to say whether there is a definite relation 
between the bacterial flora of the uterus and that of the tissues of the fetus. 
In one case, that of Tract 11, which was distinctly abnormal, the same 
bacteria were isolated from the fetal fluids as were recovered from the 
ovaries and oviducts of the dam. 

In comparing the bacteriology of aborted fetvises with that of fetuses 
removed from pregnant uteri, there is found a much larger number of 
bacteria associated with the tissues of the former. It seems strange to 
find, in checking up thft cultures, that streptococci have not been found in 
conjunction with Bacterium abortum; Staph}jlococcu^ albu^ and aureus 
have been associated with the latter many times. Fetus 32 gave pure cultures 
of a Streptococcus viridams directly from the tissues, but when extracts of 
these tissues were ground up and injected into guinea pigs, Bacterium 
abortvm was recovered from the tissues of the latter. The distribution of 
the bacteria in the fetus is remarkable. In some cases cultures of an organ- 
ism can be recovered only from the stomach contents, intestinal tracts and 
possibly the lungs, while in other cases they are recovered only from the 
heart's blood, spleen, liver, etc. At times bacteria are recovered from all 
the tissues. It seems that the sources of infection must be different in such 
cases. Evidently, when the infection is in the stomach contents, intestinal 
tracts or lungs, it must have entered the mouth of the fetus and have been 
taken in with the fetal fluids which are being swallowed continually. Where 
it is found only in the heart's blood, spleen, etc., it must have come directly 
into tlie blood stream from the cotyledon which may have been infected to such 
an extent that the epithelium has been damaged and the bacteria carried 
into the fetal circulation. When the epithelium of the intestine is damaged 
from toxins or other agents that are capable of destroying the mucous mem- 
brane, bacteria gain entrance into the circulation, as for example, the 
Bacillus tifplwsufi in typhoid fever. 

According to the data that McFadyean and Stockman (38) submit in their 
work on Epizootic Abortion, they have not found Bacterium abortum in the 
circulation of the fetus imless it was first present in the intestinal tract, 
showing that the infection was taken into the circulation from the intestinal 
tract. In several cases I have recovered bacteria from the fetal circulation 
and have been unable to get groAvths from the intestinal tract. The original 
source of the fetal fluids must be the blood stream, but there is apparently 
some difFerence of opinion as to what tissues excrete or secrete them. They 
may have two sources or more. The entrance of infection into the fetal 
fluids also seems to be in doubt. The amniotic fluid which is secreted by 
the ectoderm of the amnion is the only fluid that the fetus can take into 
its digestive tract. Therefore, infection must be present at the time of con- 
ception or within three or four days from that time if it gains access to the 
amniotic fluid. The only other possible source of infection of the amniotic 
fluid is the blood supply of the amnion. The kidney of the fetus secretes the 
allantoic fluid which becomes enormous in amount in the latter period of 
pregnancy but this does not communicate with the amniotic fluid. Do the 
bacteria come from the blood through whatever channel the fetal fluids 
pass, or are they present in the uterine wall or cavity before conception, 
having been carried there by the blood some time previous? Hagan (39) 



16 

suggests that the infection comes from the utero-chorionic space and is 
absorbed or grows into the fetal fluids through the necrotic tip of the 
chorion which is so common a condition in cattle. When infection is found 
in the genital tract of the fetris it must have been carried there by the 
circulatory system. It cannot be stated whether there are certain bacteria 
which have a sjjecial preference for the genital canal, or whether these bacteria 
become deposited there accidentally through some special arrangement of the 
tissues, and find the tract a favorable place in which to remain dormant or 
to multiply. 

The source of infection in the genital canal of adult animals presents 
more difficulties than it does in the fetus or immature animal, where, with 
perhaps the exception of the posterior part of the vagina, the external source 
can be eliminated. The two chief reasons for this are the absence of coitus 
in the yoimg and the mechanical obstruction which the tough mucus of the 
immature tract must aft'ord to bacteria. Although microorganisms have 
been isolated from only a few of the genital tracts of fetuses, calves and 
virgin heifers, a comparatively small number of such animals have been 
worked upon. It seems evident that a certain percentage carry infection in 
their genital canals from calfhood to puberty ana that this infection is borne 
there either by the blood or lymph stream. Probalrly there are certain 
individuals that are able to eliminate this infection after it is once estab- 
lished. There is also undoubtedly a group of adults that incur the infection 
during pregnancy or afterwards, in fact it is not im])ossible for the organism 
to enter the reproductive organs at any time. Rosenow has shown that 
certain streptococci, especially the viridwns group isolated from various 
sources, such as endocarditis, arthritis, duodenal ulcers, and ovaritis, have 
an affinity for these same tissues when they are injected into experimental 
animals. Detwiler and Maitland (40) found that these organisms produced 
the most lesions in the heart and joints regardless of the site of origin of the 
culture. 

There is strong evidence to prove that the streptococci which liave been 
isolated from non-breeders whose genital tracts show lesions, are the cause 
of these lesions. Rosenow and Davis (41) also have isolated Strentococois 
viridans from a large number of ovaries of women and have produced ovaritis 
in experimental animals. They report a case of a yoting girl who had a 
chronic tubo-ovarian inflammation and also a congenital stenosis of the 
cervix and uterus. They isolated a Streptococcus viridans from this case. 
Weiner (42) also reports a case of an 18 year old girl with an ovarian 
abscess and a comnlete stenosis of either the cervix or vatrina. He cultivated 
a hemolytic streptococcus from the abscess. In such cases as the last two 
quoted the source miist be hematogenic since infection by exfension is elim- 
inated. The case of Experimental Calf 10, which I have mentioned before as 
receiving an intra-uterine injection of Streptococcus viridans. followed by the 
characteristic lesions associated with this group of bacteria, supports the 
pathogenesis of the streptococcus. 

In a recent paper on Salvivgitis (43), we have shown the relation of the 
streptococci to the tubes. They were isolated in many cases of hydrosalpinx 
and other types of salpingitis. In several instances oviducts, apparently 
normal macroscopically, showed a catarrhal inflammation or a chronic pro- 
ductive inflammation on section, while streptococci were recovered from the 
lumen. 

In the case of several small aborted fetuses (30 to 4.5 days) and in one 
case of impending abortion (Pregnant Uterus 11), Streptococcus liemolyticus 
and Streptococcus viridatK'i were recovered. It does not seem possible that 
these organisms can invade tlip embrvnnic or fetal tissue without cRn«inor 
«ome damage. It may be that the streptococcus iniures the endometrium 
to such an extent that the einbryo cannot remain attached to the mucous 
membrane. If such is the case, and T have recovered streptococci from the 
uterine wall of manv cases of endometritis where the mucous membrane 
has become entirely denuded, there is no doubt that many cows which are 
considered sterile are expelling embryos that are unobserved, because the 



17 

latter cannot become implanted in the mucosa of the uterus after fertiliza- 
tion has taken place and they have descended the tube. It seems very prob- 
able that streptococci which are associated with tlie genital tracts of cows 
are pathogenic and distinctly so. In the past tliey have been regarded by 
many as saprophytes, especially the viridans group, but it seems that this 
predominating group of organisms in the genital canal will have to be 
considered more seriously if the diseases of the reproductive organs are to 
be controlled. 

There appears to be no doubt regarding the pathogenesis of most strains 
of Bwctcritim abortum. Much work has been done on natural outbreaks of 
abortion due to this organism, and also experimentally. All of the prema- 
ture exj^ulsions of fetuses are by no means due to the Bang organism. In 
the group of aborted fetuses which I have studied, I have found Bacterium 
abortum in 40 per cent, but if this disease is primarily a disease of the fetal 
membranes, perhaps a safe percentage basis cannot be established upon 
the bacteriological findings from the tissues of the fetuses. The placentae 
have been cultured in many cases and these should be examined in order 
to eliminate the presence of Bacterium, abortum,. The recovering of Bacterium 
a^bortiom in the uteri of aborted fetuses and the udders of two veal calves 
and one sterile four-year old heifer should be noted. The persistence of 
Bacterium abortum in the uterus of a calf sixty-five days after the injection 
is also interesting. The length of time that this organism remains in a non- 
pregnant genital tract has been reported to be comparatively short. Most 
investigators insist upon working with Bacterium abortum and the prema- 
ture expulsion of the fetus, but the whole problem of the diseases of the 
reproductive organs is so serious and so complicated that it cannot be solved 
by studying one organism and one condition. As the work progresses more 
scientifically and new factors are found, it is very likely that the percentage 
of abortion due to Bacterium abortum will become smaller. 

The pathogenesis of the stai)hylococci and other organisms which have been 
recovered has not been carefully studied. Their virulence may increase at 
times or the resistance of the animal become so low that they have an 
opportunity to produce disease, but they probably have little bearing upon 
the reproductive organs of a healthy individual. When they do cause disease 
they have just as important a bearing upon that particular case as would 
Bacterium abortum. or Streptococcus viridans. 

A careful search has been made for the spirillum, or vibrio, which Smith 
recovered from aborted fetuses and described. I found this organism in large 
numbers associated with an outbreak of abortion in ewes in 1919 (44), 
but I have not recovered it in connection with my work on cattle. 

One of the unexpected results of this investigation has been the compara- 
tive failure to isolate members of the colon group. These organisms are so 
easy of cultivation that, if they had been present, they would have been 
recovered. Except for cultures that have been isolated from the intestinal 
tracts of calves and fetiises that lived for only a short time, I have obtained 
only five cultures of these organisms and two of these were isolated from 
vaginae. Judging from the infrequent occurrence of this group of bacteria, 
it cannot have much weight in the etiology of intra-uterine infections of young 
calves. In the past much work has been done on the relation of the colon 
groiip to the so-called " white scours " and to different forms of calf pneu- 
monia, but I have found this organism in a verv limited number of cases 
if the calf was killed and cultured before death from the infection had 
occurred. It would appear that just before death, or at a time when the 
cells lining the intestines become injured, the large volume of infection 
normally found in the intestinal tract breaks throusrh into the circulation. 
In describing this condition, Zinsser (46) savs: "Thus, as Bail has pointed 
out, a mass of frogs' eggs will remain entirelv uninvaded while alive, though 
the water surrounding it may swarm with bacteria of many varieties, but 
when by some accident such a mass of eggs ceases to live, it immediately 
falls prey to bacterial infection. The same point is illustrated by the rapid- 
ity with which intestinal bacteria will spread through"but the body after 



18 

death, when during life, they have remained confined to the lumen of the 
intestines, or, at most, get into the portal circulation, to be destroyed in the 
liver. By the living cell, therefore, an opposition is offered to invasion by 
bacteria, a vital function which Bail has attempted to make clearer by formu- 
lating it as a law, referring to it as 'Das Gesetz Lebensundurchdringlichkeit.' " 

I have tried with many of these colon cultures and by many different 
nietiiods tu produce the same symptoms in calves but have failed in every 
attempt. .Some hold that this colon group is tlie cause of many of the calf 
infections because a serum immunized to different members of the colon 
aerogenes group of bacteria apparently has some beneficial effect upon the 
course of these maladies. In a discussion of this subject, Professor W. A. 
Hagan of the New York State Veterinary College at Cornell University sug- 
gested tliat the beneficial effect of this serum may be due to the foreign 
protein that is introduced and not to the antibodies formed in this prepara- 
tion. Because of this, normal serum has been prepared from cows, veal 
calves, and horses. In some cases whole blood has been used and in other 
cases, defibrinated blood. The results of this work have proven favorable, 
that is, in lierds where the normal serum or blood has been checked against 
the immunized serum, equally as good results have been obtained, and in 
many cases the calves on normal serum liave recovered more quickly and 
have been more vigorous than those on immunized serum. Therefore, I 
fail to see anything specific in the immunized serum that renders it of more 
vahie in treating these conditions than the normal serum. 

In three recent outbreaks of these infections in calves, I was unable to 
isolate the colon organism if the calves were killed and ctiltured a short 
time before death seemed imminent, or even immediately after death. The 
contents from the stomachs of the live calves yielded a streptococcus. The 
meconium and blood cultures and also the placentae from the dams gave 
tliis same organism in pure culture. In these herds cultures from calves 
tJiat were expelled dead gave an organism identical with that recovered 
from the living calves. In many cases I failed entirely to get cultures from 
the cadaver even though the calf had been dead some time. 

There is one type of calf infection that is due apparently to unclean food 
and insanitary surroundings. This is a form of diarrhea and it can be 
controlled easily by sanitary methods and by disinfecting the navel cord. 
The colon-aerogenes group may play a part in this type of infection but I 
do not consider it the primary causative agent. 

The cause of the intra-uterine diarrhea is apparently the infection that is 
juesent in the uterus and fetus. It is difficult to find tlie same organisms 
in a calf that has lived for a time and then died from enteritis or pneu- 
monia, that have been recovered from its dam. One cannot tell whether 
the organisms recovered from the dead calf have remained in the tissues 
since birth or whether they were picked up from some external source. With 
the exception of the instances mentioned above, I have not, as a rule, recov- 
ered the same organisms from a calf that has suffered from septicemia, 
enteritis or pneumonia that I have isolated from the uteri of pregnant ani- 
mals, though at no time have I been able to secure for bacteriological exam- 
ination a pregnant animal in a herd that was having heavy losses from these 
calf infections. There does not seem to lie enough evidence, except clinical 
evidence, to prov^ that the bacteria found in the pregnant uterus are the 
etiological factors concerned in the diseases of the young calf after birth. 

I am greatly indebted in this investigation to Professor W. L. Williams for 
his valuable suggestions and for the large amount of material obtained from 
bis department without which much of the work could not have been done. 
I am iTidebtod to Professor D. H. l^dall of the Department of Medicine and the 
ambulatory clinic for supplying me with much material for study. I am 
also indebted to Professor W. A. Hagan of the Department of Bacteriology, 
to Profess(ir J. IST. Frost of the Department of Surgery, and to Dr. H. L. 
rJilman of the Department of Obstetrics, for generous assistance. 



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18 normal genital tracts 

17 diseased genital tracts from one herd 

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herds . , 



Graph showing distribution of bacteria in genital tracts of 18 normal 
cows, 17 sterile cows slaughtered in one herd and 18 sterile cows from 
various other herds. Figures represent the number of times bacteria were 
isolated from an organ. 



19 

Bibliography 

1. Williams, W. L. Cornell Vet. 1919, 9, 204. 

2. Rei^ort of U. S. Sec'y Agriculture, 1915. 

;}. Livestock on Farms and Kanches. J.A.V.M.A., 1921, 11, 750. 

4. Dcklerlein. Centralblatt fiir Gynlik., 1894, 18, 10. 

5. Williams. Am. Jour, of the Med. Sci., 1893. 
(j. Krimig. Centralblatt f. Gyniik., 1894, 18, o. 

7. Winter. Zeit. f. Gynak., 1888, XIV, 443. 

8. Bensis. Paris Theses. 1899-1900. 

9. Bergholm. Archiv. f. Gyniik., 1902. (>(j, 497. 

''^ Walton and Medalia. ,Siirg. Gyn. and Ohs., 1912, 15, 682. 
Kiister. Kollc und Wassernvann, 1912, VI, 458. 
Permar. Am. Jour, of Obs., 1917, 75, 652. 
Rosenow. J.A.M.A., 1915, LXV, 1687. 
Davis, C. H. Surg., Gyn. and Obs., 1910, 2:;, 5(J0. 
Miller. Am. Jour, of Obs., 1916, 74, 450. 
Grad. Am. Jour, of Obs., 1918, LXXVII. 

urtis, A. X. Surg., Gyn. and Obs.. XX\'l, 1 7S. 

weedy, E. H. Surg., Gyn. and Obs., XXVll, .503. 

enzler. Inaugural Dissertation zur Krlangung der Dukterwiirde der 

liohen Vetcrinarmedizinsclien i"'akultat der Cniversitiit Zurich, 1901. 

iltncr and Bandcen. J.A.V.:\I.A., 1920, LVIl, 47. 

on Reuss. Krankliciten der Neugeboroncn, 1914. 
^. jL:,.neise. Hegar"s Bcitriige, 1902. 

23. Knapp. Monats. f. Geburts. u. Gyniik. 1897, V. 

24. Schmidgall, G. Hegar"s Beitrage." 191.3. XiX, 190. 

25. Brailowsky-Lounkevitch. Ann. de I'lnstitut Pasteur, 1915, XXIX, 379. 

26. Smith, R.M. Med. Record, Sept. 2, 1916. 

27. Hymanson and Hertz. Am. Jour, of Obs., 1917. 75, 662. 

28. Bang. Jour. Comp. Path, and Therap., 1897, 10, 125. 

29. Smith, T. Jour. Exp. Med., 1918, XXVIIl, 701. 

30. Rosenow. J.A.M.A., 1914, LXIII, 903. 

31. Smillie, Jour. Exp. :\[ed., 1918, XXVIIl. Xo. 5. 

32. Liborius. Zeit. f. Hygiene. 1886, I, 115. 

33. Nowak. Ann. de 1' Inst. Pasteur, 1908, XXII, 541. 
Wright, J. Boston Soc. Med. Sci., 1900, V, 114. 

Loeffler. Mittheilungen aus dem K. Gesundheitsamte, 1894, II. 

Huntoon, ¥. M. Jour. Inf. Dis., 1918, 23, 169. 

Brown. Monograph of the Rockefeller Institute for Med. Res., 1919, IX. 

38. McEadyean and Stockman. Rep. uf Com. App. by the Board of A"-r. 

and Fish, to inquire into Ep. Abortion. 1909. 

39. Hagan, W. A. Cornell Vet., 1917, 7, 263. 

40. Detwiler and Maitland. Jour. Exp. Med., 1918. 27, 37. 

41. Rosenow and Davis. J.A.M.A., 1916, LXVI, 1175. 

42. Weiner. Surg.. Gyn. and Obs., 1918. 27, 622. 

Carpenter, Williams and Oilman. J.A.V.M.A.. 1921. 12, 173. 
Carpenter. Cornell Vet., 1919, IX, 191. 
Zinsser. Infection and Resistance, 1914, 5. 

EXPLAXATIOX OF CHARTS 

C liart I. Pregnant uteri of cows. 

Chart II. X'ormal non-pregnant uteri of cows. 

Chart III. Diseased non-pregnant uteri of cows. 

Chart IV. Uteri from virgin heifers. 

Chart V. Uteri from calves 3-12 weeks old. 

Chart VI. Uteri from calves 1-3 weeks old. 



20 

Chart VII. Fetuses removed from pregnant uteri. 

Chart VIII. Aborted fetuses. 

Chart IX. Aborted fetuses whose dams were injected intravounisly witli 

Bact. abort II in. 
Chart X. Fetuses removed from uteri of cows sutlering from dystocia. 
Chart XI. Mummified fetuses. 
Chart XII. Experimental work. 

Blank spaces indicate negative results. 

Spaces witli — — indicate no cultures were made. 



21 



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Calf scoured for 4 days after first 
injection 12/23/20. Spleen shght- 
ly enlarged. Apparently normal 
when slaughtered. 












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PLATE I 




A. (Jiiiiu'U |)i,i; rasti'iu'il tn lioard I'v iiicaiis iif slanted nail points wliicli 
Iticrcc (he Icus. N'isrcra arc ('\|i<is('.l I'or examination. 








]j. Board, 8l^" x G" x 1", uiion wliieh <j;iiiiK'a pigs were fastened after 
lieiny killed witli eas. 






7 





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PLATE III 




Testes of tiro guinea pigs injected respectively toith extracts from Fetus 
62 and placental extracts. 1 and 2. G.. P. 2i96.— Weijjht 662 gins. 1. Shows 
tliat testicular tissiie has atropliied wiiih* tlie epididymis is eiihirged and 
eontains al)S('esses. 2. Right testicle and ei)i(lidyniis are normal. Slanghtered 
t.") days after injection. Bact. ahortum recovered t'loni epididymis of 1. 
3. (_;. P. 234. — Weight (idO gms., testicle normal. Epididymis contains 
abscesses from which lUict. ahortum was lectjvered. Slaughtered 41 days 
after injection. 



PLATE IV 




Disoari'd iiciiital tract, iiuniln'r (i7. sli(i\\iiig- ovaritis, pavilioiiit is, liydio 
salf)iiix and perinietriti.s. This trad is typical of those trnin whicii 
■;trop1o<MH'ci lia\(' lieoii isolated. 



PLATE V 




A. Cross sections of cystic right ovary of niinilier (i6 from wliicli a 
lieniolytic streptococcus (Beta) was isolated in pure culture. B. Cross 
sections of enil)e(lde(l corpus luteuni in left ovary of number 67 from 
which a streptococcus "alpha prime" was isolatcil in pure culture. 



ttATE VI 




A. Genital tract of Experimental Calf 10 whidi was 
injefted with Streptococcus riridaiis from the genita.1 
tract of a stexile cow. A. INIass of fat adherent to horns 
of uterus. B. Uterine liorns. C. Body of uterus. D. 
Ovary and |>a\ili(iii adliercnt in mass to left uterine horn. 




B. Right uterine apex, right oviduct and ovary of 
Pregnant Uterus 11, pregnant 29 days. Pavilion is ad- 
lierent to corpus luteuni of pregnancy and other areas 
on the capsule of the ovaay. The tube is cystic. Ad- 
hesions have formed since ovulation. A. Right uterine 
aipex. B. Endometrium (incision has been made in wall 
of uterine horn). C. Pavilion adherent to corpus 
luteum of pregnancy. D. Corpus luteum of pregnancy. 
E. Cystic tube. 



I'J.ATE \1I 





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A. Genital tract, 8 cm. long, of Fetus 46, showing flic Viiuiii;! 
greatly distended with nuicus. x 4. 

A. Right ovarv. B. Body of iitenis. C. ^'a^'ina. 




B. Smear made from 24 lir. agar culture of a Slrepfococ- 
<:us viridans recovered from ovaries and adhesions sur- 
rounding them of diseased genital tract of niuuber 65. 












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